Method for determining oxygen concentration

ABSTRACT

A method for determining the oxygen concentration of a sample using electron spin resonance enhanced magnetic resonance imaging.

This application is a Division of nonprovisional application Ser. No.08/546,146 filed Oct. 20, 1995 and currently U.S. Pat. No. 5,765,562.

FIELD OF THE INVENTION

This invention relates to a method for determining the oxygenconcentration of a sample, for example a human or animal body, moreparticularly to the use of electron spin resonance enhanced magneticresonance imaging (OMRI) of a sample to determine its oxygenconcentration, and especially to the use of OMRI for the generation ofimages indicative of dissolved oxygen concentration in a sample.

BACKGROUND OF THE INVENTION

Oxygen plays a key role in the metabolic processes of biological systemsand many conditions may be linked to abnormal levels of oxygen in thebody. To provide a better understanding of this metabolic role and toaid clinical diagnosis, there is clearly a need to improve the means bywhich the level of oxygen in bodily tissues may be measured.

Conventional methods for determining oxygen concentrations areunsatisfactory. One such technique involves inserting a Clark electrodedirectly into a blood vessel to determine the local oxygenconcentration. Clearly such a technique is of limited scope beinginvasive and usable only locally.

Non-invasive techniques have been slow to develop and generally are notsuited to the study of tissues lying deep beneath the surface of asample.

The most well-developed and accurate method for use ex vivo is that of“spin-label oximetry” in which changes in the esr linewidth of a freeradical caused by the presence of oxygen are monitored. Such techniquesgenerally use solid phase immobilized paramagnetic species as thespin-label and thus are not suited for in vivo measurements.

Electron spin resonance enhanced MRI, referred to herein asOMRI(Overhauser MRI) but also referred to in earlier publications asESREMRI or PEDRI, is a well-established form of MRI in which enhancementof the magnetic resonance signals from which the images are generated isachieved by virtue of the dynamic nuclear polarization (the Overhausereffect) that occurs on VHF stimulation of an esr transition in aparamagnetic material, generally a persistent free radical, in thesubject under study. Magnetic resonance signal enhancement may be by afactor of a hundred or more thus allowing OMRI images to be generatedrapidly and with relatively low primary magnetic fields.

OMRI techniques have been described by several authors, notablyLeunbach, Lurie, Ettinger, Grücker, Ehnholm and Sepponen, for example inEP-A-296833, EP-A-361551, WO-A-90/13047, J. Mag. Reson.76:366-370(1988), EP-A-302742, SMRM 9:619(1990), SMRM 6:24(1987), SMRM7:1094(1988), SMRM 8:329(1989), U.S. Pat. No. 4,719,425, SMRM8:816(1989), Mag. Reson. Med. 14:140-147(1990), SMRM 9:617(1990), SMRM9:612(1990), SMRM 9:121(1990), GB-A-2227095, DE-A-4042212 andGB-A-2220269.

In the basic OMRI technique, the imaging sequence involves initiallyirradiating a subject placed in a uniform magnetic field (the primaryfield B_(o)) with radiation, usually VHF radiation, of a frequencyselected to excite a narrow linewidth esr transition in a paramagneticenhancement agent which is in or has been administered to the subject.Dynamic nuclear polarization results in an increase in the populationdifference between the excited and ground nuclear spin states of theimaging nuclei, i.e. those nuclei, generally protons, which areresponsible for the magnetic resonance signals. Since MR signalintensity is proportional to this population difference, the subsequentstages of each imaging sequence, performed essentially as inconventional MRI techniques, result in larger amplitude MR signals beingdetected.

In any OMRI experiment under ambient conditions, paramagnetic oxygenwill have a finite effect on the spin system present. Generallyspeaking, this may be dismissed as a secondary effect when compared tothe primary interaction of the radical electron spin and the nuclearspin system. Nonetheless, it has been proposed that this effect may beused to determine oxygen concentration within a sample. However suchresearch has concentrated particularly on the use of nitroxide spinlabels; radicals which suffer the inherent disadvantage of having broadlinewidth esr resonances and therefore low sensitivity to the effects ofoxygen. Thus, to date, the effect of oxygen has been recognised only ina qualitative sense and any attempt to attach a quantitativesignificance to the oxygen effect has failed.

For example, Grücker et al (MRM, 34:219-225(1995)) reported a method forcalculating oxygen concentration by measuring the Overhauser effect in anitroxide radical and relating the non-linear effect of oxygen on theOverhauser Factor to its concentration. This involved taking two images,one on-resonance and one off-resonance, and using a first orderapproximation to arrive at the oxygen concentration. However, Grückerobserved that the correlation between actual and calculated oxygenconcentration was poor and therefore that the method was inherentlyinaccurate. This was attributed to the large number of parametersinvolved in the calculation.

Ehnholm (U.S. Pat. No. 5,289,125) has proposed an OMRI technique inwhich signals from a paramagnetic material are detected under at leasttwo different sets of operating parameters whereby to generate images ofvarious physical, chemical or biological parameters. While oxygentension was one of several such parameters, Ehnholm did not demonstratethe use of the technique to quantitate dissolved oxygen.

SUMMARY OF THE INVENTION

The present invention relates to a non-invasive method for determiningthe oxygen concentration of a sample. It involves manipulation of theOverhauser effect in which polarisation is dynamically transferred toprotons when an electron spin resonance transition of an administeredpersistent free radical is saturated. More specifically, the method isbased on observing and manipulating the varying enhancement of a protonsignal due to the changed saturation characteristics of a free radicalin the presence of oxygen.

Thus viewed from one aspect the present invention provides a method ofdetermining oxygen concentration in a sample, for example a human ornon-human, preferably mammalian, subject, said method comprising thefollowing steps: introducing into said sample an effective amount of aphysiologically tolerable free radical (generally a persistent radical)having an esr transition with a linewidth (measured in water at 37° C.)of less than 400 mG, preferably less than 150 mG; irradiating saidsample with radiation (generally referred to herein as VHF radiation) ofan amplitude (i.e. power) and frequency selected to stimulate anelectron spin resonance transition of said radical; detecting electronspin resonance enhanced magnetic resonance signals from said sampleunder at least first, second and third conditions, whereby under saidfirst and second conditions said radiation is of a first frequency,under said third conditions said radiation is of a second frequencydifferent from said first frequency, under said first, second and thirdconditions said radiation is of a first, second and third amplitude,said first and second amplitudes at least being different from eachother; and manipulating said detected signals whereby to determineoxygen concentration in said sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic example of an OMRI sequence used in the method ofthe invention.

FIG. 2 shows the linewidth as a function of oxygen concentrationmeasured at X-band for perdeuterated trityl in water and plasma.

FIG. 3 shows the linewidth as a function of oxygen concentrationmeasured at X-band for non-deuterated hydroxy trityl in water andplasma.

FIG. 4 shows the oxygen sensitivity of deuterated hydroxy trityl inwater at 37° C.

FIG. 5 shows the oxygen sensitivity of deuterated hydroxy trityl inwater at 23° C.

FIG. 6 shows the oxygen sensitivity of deuterated hydroxy trityl inblood at 37° C.

FIG. 7 shows the oxygen sensitivity of deuterated hydroxy trityl inblood at 23° C.

FIG. 8 shows the radical concentration and oxygen images calculated inblood samples at three different radical doses.

FIG. 9 shows three rat images showing in vivo oxygen concentration afterinhalation of gas of varying oxygen content.

FIG. 10 shows five rat images showing in vivo oxygen concentration afterinhalation of gas of varying oxygen content.

FIG. 11 shows five rat images showing the correlation between measuredoxygen tension and the oxygen content in an inhaled gas.

FIG. 12 shows one high power image and one calculated oxygen image of arat after clamping.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a preferred embodiment, the method of the invention comprises:

(a) introducing the radical, e.g. parenterally, for example by injectioninto body tissue or into the vasculature;

(b) generating a first OMRI image of said sample at VHF power P_(A),irradiation period T_(VHF1) and on-resonance (ΔH=O) (i.e. where thefrequency of the radiation is selected to be the resonance frequency ofthe esr transition);

(c) generating a second OMRI image of said sample at a second VHF powerP_(B), irradiation time T_(VHF1) and on-resonance (ΔH=O);

(d) generating a third OMRI image of said sample at VHF power P_(C) (egequal to P_(A) or P_(B)), irradiation time T_(VHF1) and off-resonance(ΔH≠O, for example 100-200 mG);

(e) manipulating the images obtained in steps (b) to (d) and calibratingusing parameters determined ex vivo to provide an oxygen image of saidsample.

In an especially preferred embodiment, a fourth and fifth OMRI image areadditionally generated in the imaging sequence. The conditions for thefourth image are identical to the first image but the VHF irradiationtime T_(VHF2) is different (for example twice as long, i.e.T_(VHF2)=2T_(VHF1)) and the fifth image is obtained without VHFirradiation, e.g. is a native image of intensity I_(o), generated byconventional MRI with a repetition time T_(R)=T_(VHF).

In a further embodiment, a native image (i.e. one obtained byconventional MRI) of the sample (e.g. body) may be generated to providestructural (e.g. anatomical) information upon which the oxygen image maybe superimposed. In this way, precise location of for example an oxygendeficient tumour will be possible.

Accurate measurement of the level of oxygen in bodily tissues is aninvaluable aid to the clinician and the method of the invention has avariety of end uses. For example, knowledge of the concentration ofoxygen dissolved in blood can be used (through known rate constants) tocalculate the concentration of oxygen associated with haemoglobin. Thisis a useful parameter which is presently measured either by undesirableinvasive techniques or using the BOLD MR imaging technique whichinvolves high field imaging to determine the effect of oxygen onparamagnetic iron but which has the disadvantage that to determine bloodoxygen concentration the volume of blood in which the measurement wasmade needs to be known.

Other uses of the method of the invention will be readily apparent tothe skilled person and include oxygen imaging (e.g. mapping) of, forexample, the heart and arteries and of malignant tumours, for example inthe brain, breast, lung, lymphoid tissues and superficial areas of theliver. In the case of oxygen imaging of tumours, success in treatment ofmalignant tumours by radiotherapy may be reflected in the level ofoxygen in the tissue (typically an oxygen concentration of less than0.01 mM will indicate that the tissue is necrotic and thus thattreatment is likely to be ineffective).

It will also be apparent that the method of the invention will be usefulin cardiology, surgery and intensive care where levels of oxygen andeven perfusion can be non-invasively assessed in almost any tissue.

The manipulation of the detected MR signals in the method of theinvention will generally be to generate an image data set (i.e. a dataset from which an image may be generated) indicative of radicalconcentration and one or more image data sets indicative of radicalelectron relaxation times (generally T_(1e), T_(2e) or T_(1e).T_(2e))and manipulation of these data sets and calibration with ex vivocalibration data to yield an image data set indicative of oxygenconcentration. This oxygen concentration image data set can betransformed into an oxygen concentration image or can be subject to anupper or lower limit filter to identify regions of high or low oxygenconcentration, which can again if desired be displayed as an image.

Broadly speaking, the Overhauser enhancement of the proton MR signal isdependent on the relaxation times T_(1e) and T_(2e) of the esrtransition of the radical used in the method of the invention. Theserelaxation times themselves are dependent on the concentrations of theradical and dissolved oxygen in the body fluid as well as on thetemperature and chemical nature of the body fluid. However while theOverhauser enhancement can easily be used to determine the oxygenconcentration for an isolated small volume sample of known radicalconcentration ex vivo, the determination of oxygen concentration in vivois complicated since the Overhauser enhancement is also stronglydependent on the sample structure for a large non-isolated sample, suchas a living body, due inter alia to non-uniform radiation penetrationinto the large sample.

Thus although the method of the invention requires calibration data,obtained for a range of radical and oxygen concentrations in a fluidsample (e.g. blood) which corresponds to the body fluid in whichoxygenation is to be determined and at a pre-set temperature (e.g. 37°C.), further data manipulation is required in order to extract the invivo oxygen concentrations from the OMRI signals detected for thesample.

The calibration data are generated by determining Overhauser enhancementvalues for the radical in the selected body fluid, at the selectedtemperature and at a range of oxygen (and preferably also radical)concentrations. The intrinsic esr relaxation times for the radical canbe determined, under the same conditions, using a conventional esrspectrometer equipped with a temperature controller, with oxygenconcentration being determined using the method of Ravin et al J. Appl.Physiol. 18:784-790(1964), a method known to produce accurate andreproducible results.

In general, radical concentrations up to 0.2, preferably up to 1.0,especially up to 1.5 mM, and oxygen concentrations of up to 0.1,preferably up to 0.5 mM should be investigated to generate thecalibration data.

For one preferred radical, referred to herein as the perdeuteratedhydroxy trityl, such calibration of a blood sample at 37° C. showedmaximum Overhauser enhancement (i.e. at infinite VHF power and infiniteradical concentration) to be 192 and T₁ i.e. proton relaxivity to be0.44 mM⁻¹s⁻¹. The dependence of T_(1e) and T_(2e) on radical and oxygenconcentrations was found to fit the following linear functions:$\begin{matrix}{{2\left( {\sqrt{3}\gamma_{e}T_{2e}} \right)^{- 1}} = {20 + {21.5\quad C_{rad}} + {428\quad C_{O_{2}}}}} & (1) \\{{2\left( {\sqrt{3}\gamma_{e}T_{1e}} \right)^{- 1}} = {10 + {3.6\quad C_{rad}} + {330\quad C_{O_{2}}}}} & (2) \\{\left( {\gamma_{e}\sqrt{T_{1e}T_{2e}}} \right)^{- 1} = {15 + {6.9\quad C_{rad}} + {319\quad C_{O_{2}}}}} & (3)\end{matrix}$

where γ_(e) is the electron gyromagnetic ratio, C_(rad) is the radicalconcentration in mM, C_(o) ₂ is the oxygen concentration in mM andT_(1e) and T_(2e) are electron relaxation times in s, and coefficientsare in mG, the units in which linewidth is measured.

Similar equations can be derived experimentally for whatever radical isused in the method of the invention.

With this calibration data, if T_(1e), T_(2e) or T_(1e).T_(2e) arecalculated for a pixel in the sample's OMRI image then equations (1),(2) or (3) can easily be used to determine the oxygen concentration forthat pixel. The radical concentration can be determined by manipulationof the MR signals detected in the method of the invention whereby togenerate a radical concentration image data set.

However, the T_(1e), T_(2e) or T_(1e).T_(2e) values for the pixel mustbe extracted from the OMRI signals detected in the imaging procedure.The OMRI imaging sequence used in the method of the invention may be anyone of the conventional sequences. However an example of one such usablesequence is shown schematically in FIG. 1. This sequence involves a VHFirradiation period (T_(VHF)) of approximately the same magnitude as T₁for the water proton, and a single echo of time TE much less than T₂.Pixel intensity (I) is then given by equation (4):

Iα(1−exp(−T_(VHF)/T₁))  (4)

During VHF irradiation, dynamic proton polarization <I_(z)> occurs. Thesteady state is governed by the Overhauser equation (5) $\begin{matrix}{\frac{\langle I_{z}\rangle}{I_{o}} = {1 - \left\lbrack {\frac{S_{o}{k \cdot f}}{I_{o}} \cdot \frac{S_{o} - {\langle S_{z}\rangle}}{S_{o}}} \right\rbrack}} & (5)\end{matrix}$

where $\frac{S_{o}}{I_{o}}$

 is equal to 658 for an electron: proton dynamic nuclear polarization(I_(o) here represents the equilibrium magnetisation),

k is the coupling factor (equal to ½ at low field),

f is the leakage factor,

and (S_(o)−<S_(z)>)/S_(o) is the degree of saturation (SAT) of theelectron spin transition).

The leakage factor f is given by equation (6) $\begin{matrix}{f = {\frac{{rC}_{rad}T_{10}}{1 + {{rC}_{rad}T_{10}}} = {rcT}_{10}}} & (6)\end{matrix}$

(where

r is the relaxivity of the radical;

C_(rad) is the radical concentration, and

T₁₀ is the proton relaxation time T₁ in the absence of the radical).

The pixel intensity of the final image is given by equation (7)

Iα(1−exp(−T_(VHF)/T₁))(1−329rC_(rad)T₁SAT)I_(o)  (7)

(where

I_(o) is the intensity of the native image pixel)

As can be seen from a Taylorian expansion of the exponential function inequation (7), provided that T_(VHF) is significantly less than T₁₀, T₁disappears to a first order. SAT depends on the strength of the excitingVHF field B_(1e) and obeys the basic Bloch equations. Where the esrtransition is a single Lorentzian this means that SAT is given byequation (8) $\begin{matrix}{{SAT} = \frac{\alpha \quad P\quad \gamma_{e}^{2}T_{1e}T_{2e}}{1 + {\alpha \quad P\quad \gamma_{e}^{2}T_{1e}T_{2e}} + \left( {\Delta \quad \omega \quad T_{2e}} \right)^{2}}} & (8)\end{matrix}$

(where

α is a conversion factor;

P is the VHF power; and

Δω is the distance from resonance of the off-resonance VHF excitationfrequency (where an on-resonance frequency is used, Δω is of coursezero)).

The conversion factor α is strongly spatially variant in in vivo largesample images, and thus knowledge of P, SAT, γ_(e) and Δω is not initself sufficient to enable oxygen concentration to be determined.

In most cases, moreover, the esr transition will not be a singleLorentzian due to residual magnetic couplings within the radicalmolecule. Where, as in the case with narrow esr linewidth radicals suchas the trityls mentioned herein, the coupling constants are much smallerthan the linewidth, the resonance lineshape will become a Voigt functionand SAT will be the integral of all off-resonance values weighted by aGaussian intensity function as in equation (9) $\begin{matrix}{{SAT} = {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{\Delta \quad H_{pp}^{G}}{\int_{- \infty}^{\infty}{{\exp \left( {{- 2}{H^{\prime 2}/\Delta}\quad H_{pp}^{G^{2}}} \right)}\frac{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P\quad \gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{I}}}{H^{I}}}}}}} & (9)\end{matrix}$

(where ΔH_(PP) ^(G) and ΔH_(PP) ^(L) are the first derivativepeak-to-linewidth of the Gaussian and Lorentzian functions and in fieldunits ΔH is the off-resonance field).

Equations (8) and (9) apply to single esr peaks homogeneously orinhomogeneously broadened respectively. For well separated peaks, withlarge couplings, the saturation degree will be reduced by a factorcorresponding to the far-off-resonance fraction (⅓ for nitroxides due tonitrogen coupling and 0.8 for trityls due to multiple ¹³C couplings).

In the method of the invention, the data manipulation will in general beto fit SAT as determined on a pixel-by-pixel basis to one of equations(8) or (9) and thereby extract T_(1e), T_(2e) or T_(1e).T_(2e), again ona pixel-by-pixel basis so permitting pixel oxygen concentration to becalculated from equations (1), (2) or (3) (or the appropriate equivalentequation for the radical used in the method).

In one preferred embodiment of the method of the invention, datamanipulation is effected to calculate esr linewidth based oninhomogeneous broadening (equation (9)).

At its most elementary, this method requires three OMRI images to begenerated. These however can be and preferably are supplemented withfurther images recorded off-resonance, and also preferably aresupplemented by images recorded with different irradiation times andnative images.

In the elementary version of the method images A, B and C are recordedas follows:

A: VHF power P_(A). Δω=0 (i.e. on-resonance) ΔH=0. Irradiation timeT_(VHF)=T_(VHF1)

B: VHF power P_(B) (≠P_(A)). Δω=0. Irradiation time T_(VHF)=T_(VHF1)

C: VHF power P_(C) (e.g.=P_(A) or =P_(B)). Δω≠0 (i.e. off-resonance)ΔH≠0 (e.g. 100-200 mG). Irradiation time T_(VHF)=T_(VHF1)

Under these conditions, pixel intensity can be written as:$\begin{matrix}{I = {{A\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{\Delta \quad H_{pp}^{G}}{\int_{- \infty}^{\infty}{{\exp \left( {{- 2}{H^{\prime 2}/\Delta}\quad H_{pp}^{G^{2}}} \right)}{\frac{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P\quad \gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}} \cdot {H^{\prime}}}}}}} \right\}} - B}} & (10)\end{matrix}$

(where A=Gain×proton density×rC_(rad)T₁×(1−exp(−T_(VHF)/T₁)) forrC_(rad)T₁<<1

(Gain is the system gain factor and proton density is the proton densityof the pixel);

and B=Gain×proton density×(1−exp(−T_(VHF)/T₁))).

Equation 10 contains five unknowns: T₁, proton density, C_(rad),${\Delta \quad H_{pp}^{L}} = \frac{2}{\sqrt{3}{\gamma_{e} \cdot T_{2e}}}$

and αT_(1e).

With a large enhancement (e.g. about 10), short T_(VHF1) relative to T₁and essentially uniform proton density in the fluid medium in which theradical is distributed, B can be omitted and the three unknowns C_(rad),ΔH_(PP) ^(L) and α T_(1e) can be fitted on a pixel-by-pixel basis fromthe three values of I obtained from images A, B and C respectively.Radical concentration (C_(rad)) can then be determined by scaling A withGain and r to yield a radical concentration image. Using the determinedvalue of ΔH_(PP) ^(L) and the radical concentration image, the oxygenconcentration image can then be calculated from equation (1).

A more accurate determination of oxygen concentration can be made usingthis method if two further images are generated, one image Don-resonance, at power P_(A) and at irradiation time T_(VHF)=T_(VHF2)(where T_(VHF2)≠T_(VHF1), e.g. T_(VHF2)=×2T_(VHF1)), and the secondimage E without VHF stimulation, using conventional MR with repetitiontime TR=T_(VHF1). Image E gives the native intensity I_(o) for thepixels.

From the five values for pixel intensity all five unknowns can becalculated, again yielding a concentration image and ΔH_(PP) ^(L) fromwhich an oxygen concentration image can be determined using equation(1).

In this method, if reference samples containing body fluid and radical,are disposed about the sample surface (e.g. tubes of blood containingthe radical at known concentration), the oxygen concentration image canbe adjusted to express concentration even more accurately.

A further preferred embodiment of the method of the invention takesadvantage of the greater sensitivity to oxygen concentration of equation(3), i.e. of the product T_(1e).T_(2e). This method however requires α,which gives the VHF magnetic field at the pixel, to be determined.

In this further method, oxygen concentration and radical concentrationimages are calculated from three or more images as above, a 1/T_(1e)image is calculated from these images and an α-image is calculated bymultiplying the 1/T_(1e) image by αT_(1e) as determined. The α-image isthen smoothed using for example a polynomial function. It is preferredthat reference samples be disposed about the sample under investigationas discussed above. If this is done then the smoothing of the α imagecan be achieved using a smoothing function with fixed values at thereference sample sites. This reduces statistical error in the images, isjustified as the spatial variance of α is slow and, with fixed referencepoints, produces an accurate α image.

Using this α-image, the product of ΔH_(PP) ^(L) and 1/T_(1e) can becalculated and from this (which is dependant on 1/T_(1e).T_(2e)) and theradical concentration image, a more precise oxygen image can becalculated.

If reference sample tubes are not used, then the smoothed α-image canstill be calculated but in this event the α-values determined arepreferably used in the calculation of the three (or five) variables fromthe detected OMRI images with a further smoothed α-image beingcalculated from the resulting 1/T_(1e) image and with the procedurebeing repeated until successively generated α-images are essentiallyunchanged (i.e. the procedure converges to a best-fit).

The various methods of calculation applied to the data collected in themethod according to the invention represent a significant step forwardin the accurate determination of oxygen concentration in a sample.Whilst for radicals (typically nitroxides) having a large esr linewidththe Lorentzian model is an accurate approximation for the lineshape, inthe case of narrow esr linewidth radicals more precise analysis of thelineshape is called for and leads to a more accurate determination ofthe oxygen concentration.

Thus the method according to the invention leads to an agreement factorbetween actual and calculated oxygen concentration typically less thanor equal to about 5% for a 3×3×10 mm voxel, 100 second acquisition time,0-0.1 mM oxygen concentration and radical dosage of 0.1-0.2 mmol/kgbodyweight, for samples being typically of the size of a human body.

In allowing the spatial variation of the VHF magnetic field to becalculated, the further method described above yields an absolutequantification of the longitudinal relation time (or the product of thelongitudinal and transverse relaxation time). The longitudinalrelaxation time (and even more so the product of the longitudinal andtransverse relaxation rates) is more sensitive to oxygen and so thismethod overall is the more sensitive technique.

Although the above described methods have focused on the use of Voigtfunctions to calculate the various unknown parameters, the method of theinvention may equally involve the use of Lorentzian functions wherethese are an accurate model of the esr lineshape and such a method formsa further embodiment of the invention. For example, in large linewidthradicals (typically nitroxide radicals) the effects of inhomogeneity maybe neglected and the lineshape will essentially be Lorentzian. Thus inthis preferred embodiment, the data manipulation step will essentiallyamount to fitting SAT (as determined on a pixel-by-pixel basis) toequation (8), extracting T_(1e), T_(2e) and T_(1e).T_(2e) on apixel-by-pixel basis thereby permitting oxygen concentration to bedetermined from empirical relationships such as equations (1), (2) and(3).

In practice, it may be necessary to compensate for flow effects in themethod of the invention and the appropriate steps will be known to thoseskilled in the art. Other parameters such as for example sampleviscosity, pH, temperature, radical self-broadening, etc. are typicallyonly secondary effects and thus may be neglected when compared to thefirst order effects of paramagnetic oxygen in the method of theinvention. Radical self-broadening is however corrected for in equations1 to 3.

Generally speaking, for the present method any conventional persistentfree radical may be used provided it is stable under physiologicalconditions, has a sufficiently long half life (at least one minute,preferably at least one hour), has a long electronic relaxation time andgood relaxivity. It will be apparent from the discussion of the methodof the invention that the sensitivity of the oxygen measurement will beimproved with radicals having narrow linewidth esr transitions, e.g. upto 500 mG, preferably less than 150 mG, especially less than 60 mG. Byway of illustration, for a typical oxygen sensitivity in terms of linebroadening of 500 mG/mMO₂, a radical with a T_(2e) related linewidth of500 mG (for example nitroxide radicals of the type proposed by Lurie etal in J. Mag. Reson. 76:366-370(1988)) would give only a 10% increase inlinewidth for an increase in oxygen concentration of 0.1 mM, whereas fora radical with a linewidth of 50 mG there would be a 100% increase foran equivalent increase in oxygen concentration.

Preferably, the radical selected for use in the present method shoulddistribute substantially into the extracellular fluid (i.e. should be anECF agent) since the effects of paramagnetic iron (e.g. the iron withinthe red blood cells) may be avoided there.

Another preferred characteristic of the radicals for use in the presentmethod is that they should have a low self-broadening effect, preferablyless than 100 mG, especially preferably between 0 and 50 mG per mM ofthe radical itself.

One particularly preferred class of compounds exhibiting low esrlinewidths and self-broadening effects particularly suited to thepresent method is the triarylmethyl radicals (hereinafter referred to as“trityls”) as discussed in WO-A-91/12024, U.S. patent application Ser.No. 08/220,522 and U.S. patent application Ser. No. 08/467,273 (NycomedInnovation AB) and International Patent Application No. PCT/GB95/02151of Nycomed Imaging AS as well as their deuterated analogs.

Especially preferred trityls for use in the method of the invention arethose of formula:

wherein:

n is 0, 1, 2 or 3;

R¹ is a carboxyl group or a derivative thereof;

R² is an optionally hydroxylated C₁₋₆-alkyl group; preferably a C^(n)H₃or C^(n)H₂OH group (where n is 1 or 2 i.e. ²H is deuterium); and thesalts and precursors and deuterated analogs thereof.

Naturally, this definition is intended to cover radical precursors whichmay undergo a radical generation step shortly before administration oreven in situ to produce the free radical. Radical precursors and radicalgeneration steps are well-known to those skilled in the art. Especiallypreferred trityls are those of the following formulae (herein referredto as perdeuterated trityl, non-deuterated hydroxy trityl, deuteratedhydroxy trityl and symmetric trityl respectively):

The preparation of free radicals appropriate for use in the presentmethod is in many cases a well known synthetic procedure and in othercases is discussed for example in WO-A-91/12024, U.S. patent applicationSer. No. 08/220,522 and U.S. patent application Ser. No. 08/467,273(Nycomed Innovation AB). Perdeuterated trityl may be prepared by themethod described for the preparation of its non-deuterated analogue inExamples 15 to 20 below but with the use of acetone-d₆ instead ofacetone in the initial ketalisation step (described in Example 2 ofWO-A-91/12024). Deuterated hydroxy trityl is prepared generally bysuccessive steps of fused ring formation and deuterative reductionfollowed by analogous steps to those described for the preparation ofthe non-deuterated analogue in Examples 23 to 27 below.

The perdueterated trityl and deuterated hydroxy trityl are novelcompounds and form a further aspect of the invention.

Another particularly useful class of radical compounds for the method ofthe invention are the deuterated nitroxide radicals, especiallyperdeuterated 2,5-di-t-butyl-3,4-dimethoxycarbonyl-pyrryloxyls whichhave remarkably low linewidths. These compounds may be prepared from2,5-di-t-butyl-3,4-dimethoxycarbonyl-pyrryloxyl by deuterating themethyl ester moiety by transesterification with methanol-d₄ and/or thet-butyl groups via a multistep sequence starting from acetone-d6.

For in vivo imaging, the radical compound should of course be aphysiologically tolerable radical or one presented in a physiologicallytolerable form (e.g. in solution, encapsulated or as a precursor). Theradicals may be conveniently formulated into contrast media togetherwith conventional pharmaceutical carriers or excipients.

Contrast media used according to this invention may contain, besides theinert free radicals (or the non-radical precursor where radicalformation is to be effected immediately before administration),formulation aids such as are conventional for therapeutic and diagnosticcompositions in human or veterinary medicine. Thus the media may forexample include solubilizing agents, emulsifiers, viscosity enhancers,buffers, etc. The media may be in forms suitable for parenteral (e.g.intravenous) or enteral (e.g. oral) application, for example forapplication directly into body cavities having external voidance ducts(such as the gastrointestinal tract the bladder and the uterus), or forinjection or infusion into the cardiovascular system, muscle or othertissue. However solutions, suspension and dispersions in physiologicaltolerable media will generally be preferred.

For use in in vivo diagnostic imaging, the medium, which preferably willbe substantially isotonic, may conveniently be administered at aconcentration sufficient to yield a 1 micromolar to 10 mM preferably0.05 to 1 mM, especially 0.1 to 0.3 mM concentration of the free radicalin the imaging zone; however the precise concentration and dosage willof course depend upon a range of factors such as toxicity, the organtargeting ability of the contrast agent, and the administration route.The optimum concentration for the free radical represents a balancebetween various factors. In general, optimum concentrations would inmost cases lie in the range 0.1 to 100 mM, especially 0.2 to 10 mM, moreespecially 0.5 to 5 mM. Compositions for intravenous administrationwould preferably contain the free radical in concentrations of 1 to 1000mM especially 5 to 500 mM. For ionic materials, the concentration willparticularly preferably be in the range 5 to 200 mM, especially 10 to150 mM and for non-ionic materials 20 to 400 mM, especially 30 to 300mM.

Thus viewed from a different aspect the present invention provides theuse of persistent free radicals, preferably radicals of low intrinsicesr linewidth, particularly preferably trityl radicals, in in vivooximetry.

The following Examples are intended to illustrate the invention in annon-limiting manner.

EXAMPLES

The following four water soluble, single ESR line trityls wereinvestigated by NMRD, Dynamic Nuclear Polarisation (DNP) and ESR(Examples 1-4).

(1) Bis-(8-sodiumcarboxylate-2,2,6,6-tetrakis-(²H₃methyl)-benzo[1,2-d:4,5-d′]-bis(1,3)-dithiole-4-yl)-mono-(8-sodiumcarboxylate-2,2,6,6-tetrakis-(²H₃-methyl)-benzo[1,2-d:4,5-d′]-bis(1,3)-dioxole-4-yl)methyl

Herein referred to as perdeuterated trityl (MW=1080).

(2) Bis-(8-sodium carboxylate-2,2,6,6-tetrahydroxymethylbenzo[1,2-d:4,5-d′]-bis(1,3) dithiole-4-yl)-mono-(8-sodiumcarboxylate-2,2,6,6-tetramethylbenzo [1,2-d:4,5-d′]-bis(1,3)dioxole-4-yl) methyl

Herein referred to as non-deuterated hydroxy trityl (MW=1129).

(3) Bis-(8-sodiumcarboxylate-2,2,6,6-tetrakis-(hydroxy-²H₂-methyl)-benzo[1,2-d:4,5-d′]-bis(1,3)dithiole-4-yl)-mono-(8-sodium carboxylate-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3) dioxole-4-yl) methyl

Herein referred to as deuterated hydroxy trityl (MW=1145).

(4) Tris-(8-sodiumcarboxylate-2,2,6,6-tetrakis-(²H₃-methyl)-benzo[1,2-d:4,5-d′]-bis(1,3)dithiole)methyl

Herein referred to as symmetric trityl (MW=1151).

Example 1

The relaxivity and DNP enhancement data were measured in water, plasmaand blood at 23° C. and 37° C. The results are set out in Tables 1 and2.

TABLE 1 Parameters from the NMRD profiles and DNP enhancement curves ofthe deuterated hydroxy trityl in plasma and blood Relaxivity at infiniteA. Relaxivity concentration (infinite at 400kHz and power concentration[mM⁻¹s^(−1]) [mM⁻¹s^(−1]) and power) plasma 23° C. 0.48 37° C. 0.45 0.32231 blood 23° C. 0.53 37° C. 0.44 0.44 192

TABLE 2 Relaxivities and enhancements at infinite concentration andpower for three radicals in water Perdeuterated Deuterated Symmetrictrityl hydroxy trityl trityl Relaxivity [mM⁻¹s^(−1])  23° C. 0.19 0.260.21  37° C. 0.14 0.20 0.15 A. 267 278 266

Example 2

Electron spin relaxation rates were measured by analysis of CW ESRspectra and DNP data at 23° C. and 37° C. in water, isotonic saline,plasma and blood for the deuterated hydroxy trityl and in water andisotonic saline for the perdeuterated and symmetric trityl. The resultsare set out in Table 3:

TABLE 3 Concentration dependent relaxation rates in isotonic salinewater for three radicals in water expressed in mG/mM peak-to-peak valuesand in plasma and blood at 37° C. for the deuterated hydroxy tritylPerdeuterated Deuterated Symmetric trityl hydroxy trityl trityl water23° C. 24.3 ± 0.8 11.1 ± 0.5  35.4 ± 0.6 2({square root over (3)}γ_(e)T_(2e))⁻¹ 37° C. 28.2 ± 1.2 8.0 ± 0.2 33.1 ± 0.3 water 23° C. 10.6 ± 0.82.6 ± 0.1 13.2 ± 0.5 2({square root over (3)}γ_(e) T_(1e))⁻¹ 37° C. 12.9± 0.3 2.8 ± 0.2 17.7 ± 0.8 plasma 2({square root over (3)}γ_(e)T_(2e))⁻¹ 9.8 ± 0.5 37° C. 2({square root over (3)}γ_(e) T_(1e))⁻¹ 2.7 ±0.5 blood 2({square root over (3)}γ_(e) T_(2e))⁻¹ 21.5 ± 0.5  37° C.2({square root over (3)}γ_(e) T_(1e))⁻¹ 3.6 ± 0.5

Example 3

Oximetric calibrations were made for the perdeuterated trityl and thenon-deuterated hydroxy trityl in water and plasma at 37° C. with ESR inthe X-band at 9 GHz. The experiment was performed on a Varian X-bandspectrometer with temperature controller. A thermocouple was placed nearthe microwave cavity for accurate determination of the temperature. Thesamples were placed in thin wall teflon capillaries for rapidequilibration with a flowing gas mixture. A Sensormedics Oxygen AnalyzerOM-11 was used to determine the oxygen percentage in the flowing gas.The gas pressure for temperature control and oxygenation was maintainedat 20 psi. The linewidth and the saturation of the electron spinresonance was measured. Fremys salt was chosen as a B₁ calibrationstandard and a conversion factor of 38.6±0.2 mG/{square root over (mW)}was obtained.

The results are shown in FIGS. 2 and 3 which give the linewidth as afunction of oxygen concentration. The oxygen broadening of theperdeuterated trityl in plasma at 37° C. is 583 mG/mM_(o) ₂ .

Example 4

The oxygen sensitivity of the deuterated hydroxy trityl was examined at260 MHz in water and blood at 23° C. and 37° C. by ESR and DNP. Thedesired oxygen partial pressures were obtained in a simple shakingtonometer. The sample of 1-2 ml volume was shaken with a water saturatedgas mixture flowing slowly above the sample for 5 min. The sample andgas was in a water bath maintaining the temperature. The gas mixtureswere of high purity, chemically analysed. The results are shown in FIGS.4-7 and in Table 4. The Lorentzian line broadenings are 511 and 369mG/mM_(o) ₂ in water at 37° C. and 23° C. respectively and 329 mG/mM_(o)₂ in blood at 23° C.

TABLE 4 Relaxation rates in mG/mM peak-to-peak values and the squareroot of slope of the inverse DNP curve in mG/mM as a function of oxygenconcentrations for the deuterated hydroxy trityl at 23° C. and 37° C. inwater and blood. (γ_(e){square root over (T_(1e) +L T_(2e)}))⁻¹2({square root over (3)}γ_(e)T_(2e))⁻¹ 2({square root over(3)}γ_(e)T_(1e))⁻¹ water, 37° C. 359C_(o) ₂ + 14.2 511 398 water, 23° C.282C_(o) ₂ + 13.4 369 301 blood, 37° C. 319C_(o) ₂ + 15.0 428 330 blood,23° C. 249C_(o) ₂ + 12.9 329 250

Example 5

The following experiments were performed using the hydroxy tritylradical (as hereinbefore defined) and carried out on a Picker NordstarMEGA 4 250-300 MR machine adapted for use in OMRI by reduction of theprimary field strength from 0.1 to 0.01T and by the incorporation of aVHF emitter to emit VHF radiation in the frequency range 200 to 300 MHzand the power range 0 to 100 W.

(a) Blood Samples with Different Radical Concentrations and OxygenTension

Radical concentration and oxygen images were calculated in blood samplesat three different radical doses, 2.0 mM, 4.0 mM and 6.0 mM. The resultsare shown in FIG. 8.

Parameters:

Scan time 4:36 min TR/TE  270 ms/20 ms Slice   4 mm Pixel size 0.5 × 0.5mm² Average   2 T-vhf  200 ms Sampling time   24 ms Samp. freq.   21 kHzSamp. matrix 512 × 256 (Oversampling in read direction) Recon. matrix256 × 256

(b) OMRI Oximetry

Three images of rats weighing 120 g were obtained showing in vivo oxygenconcentration after inhalation of gas of varying oxygen content. Theradical dose was 1.5 mmol/kg injected into the tail vein in a volume of1.5 ml and an injection time of 10 s (10 s before the first image wasobtained). The results are showing in FIG. 9.

Parameters:

Scan time 3:28 min TR/TE  270 ms/20 ms Slice   5 mm Pixel size 0.75 ×0.75 mm² Average   2 T-vhf  200 ms Sampling time   24 ms Samp. freq.  21 kHz Samp. matrix 512 × 192 (Oversampling in read direction) Recon.matrix 192 × 192

(c) OMRI Oximetry

Five images of rats weighing 125 g were obtained showing in vivo oxygenconcentration after inhalation of gas of varying oxygen content. Theradical dose was 1.5 mmol/kg injected into the tail vein in a volume of1.5 ml and an injection time of 15 s (15 s before the first image wasobtained). The results are shown in FIG. 10.

Parameters:

Scan time 3:28 min TR/TE 270 ms/20 ms Slice 5 mm Pixel size 0.75 × 0.75mm² Average 2 T-vhf 200 ms Sampling time 24 ms Samp. freq. 21 kHz Samp.matrix 512 × 192 (oversampling in read direction) Recon. matix 192 × 192

(d) OMRI Oximetry

An experiment was performed to investigate the correlation betweenmeasured oxygen tension in the lungs and the oxygen content in aninhaled gas. Rats weighing 150 g were injected in the tail vein with theradical in a dose of 1.0 mmol/kg in an injection volume of 0.5 ml and aninjection time of 10 s (10 s before the first image was obtained). Theresults are shown in FIG. 11.

Parameters:

Scan time 1:44 min TR/TE  270 ms/18 ms Slice   5 mm Pixel size 1.0 × 2.0mm² Average   2 T-vhf  200 ms Sampling time   24 ms Samp. freq.   21 kHzSamp. matrix 512 × 96 (Oversampling in read direction) Recon. matrix 192× 96

(e) OMRI Oximetry

One high power image and one calculated oxygen image were obtained afterclamping. Rats weighing 132 g were injected in the tail vein with theradical in a dose of 2 mmol/kg in an injection volume of 1.0 ml and aninjection time of 60 s (15 minutes before the first image was obtained).Imaging was started 8 minutes after clamping (see FIG. 12).

Parameters:

Scan time 3:28 min TR/TE  270 ms/20 ms Slice   8 mm Pixel size 1.0 × 2.0mm² Average   2 T-vhf  200 ms Sampling time   24 ms Samp. freq.   21 kHzSamp. matrix 512 × 192 (Oversampling in read direction) Recon. matrix192 × 192

Example 6

The ESR-spectral properties of a series of partially and fullydeuterated nitroxides (compounds 3b-d) also useful in the method of theinvention have been investigated. These radicals are derived from2,5-di-t-butyl-3,4-dimethoxycarbonyl-pyrryloxyl (Compound 3a) and wereinvestigated alongside Tempone(4-oxo-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1) and CTPO(3-carbamoyl-2,2,5,5-tetramethyl-pyrroline-1-yloxyl, 2a) which aretypical examples of nitroxides frequently used for imaging purposes.

Materials. 4-oxo-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1 (Jansen, 95%),4-oxo-2,2,6,6-tetramethyl-piperidine-d₁₆-1-oxyl, 1-d₁₆ (MSD isotopes, 98atom-% D) and 2,2,5,5-tetramethyl-3-pyrrolin-d₁₃-oxyl-3-carboxylic acid,2b-d₁₃ (MSD isotopes, 97.5 atom-% D) were used as received.2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid, 2b, wasavailable from earlier work. Methyl-4,4-dimethyl-3-oxo-pentanoate(Aldrich, 99%) (5a), trimethylsilyl iodide (TMSI, Jansen, 97%),acetone-d₆ (Glaser AG, >99.5% D) and methanol-d₄ (CIL, >99.8% D) wereused as supplied. Pinacolone-d₁₂ ws prepared from acetone-d₆ asdescribed in Organic Synthesis Coll., 1, 459-462 for the non-deuteradedcompounds. Diethyl ether (Anhydroscan <0.01% H₂O) was passed throughneutral alumina prior to use. Sodium hydride (Aldrich, 80% suspension inmineral oil) and nickel peroxide (Aldrich) were used as received. Allother chemicals were of highest commercial quality available and used assupplied.

Instrumentation. The ESR-spectra were recorded by the Upgrade VersionESP 3220-200SH of a Bruker ER-200D SRC instrument at 22°. The radicalconcentration was in the range 0.1-0.2 mM and the modulation amplitudewas 10 mG. The microwave power was well below saturation. NMR-spectrawere recorded on a Varian XL-300 spectrometer. Mass spectra wererecorded on a VG Quattor II instrument equipped with ESPC electrospray.GLC analyses were performed on a HP 5830 ser II instrument, equippedwith a fused-silica column (30 m, 0.25 μm, HP-1701). TLC analyses andcolumn chromotographic separations were performed on Silica Gel 60,using heptane/ether as the eluent.

Preparation of 5c. NaH (22.9 g, 0.77 mol) and dimethyl carbonate (64.1g, 0.77 mol) were treated with pinacolone-d₁₂ (32.4 g, 0.29 mol) asdescribed for the non-deuterated compound in J. Am. Chem. Soc., 72, 1356(1950) to give 5c (28.1 g, 0.17 mol, 59%) boiling at 98-100° C./6 mm. ¹HNMR (CD₃OD): δ 3.72 (s, 3H). ¹³C NMR (CD₃OD): δ 209.0 (—CO—), 168.8—COO—), 51.2 (—CH₃), 43.7-42.0 (m, —CD₂), 25.0-23.5 (m, —CD₃).

Preparation of 5b and 5d. The methyl-h₃ ester (5a, 5c) was dissolved inCD₃OD and treated with 2 mol-% NaOCD₃. After evaporation the procedurewas repeated, and when no protons from the methyl group were discernibleby NMR the transesterification was judged to be complete. The mixturewas evaporated, ether added, evaporated and used without furtherpurification in the next step.

Preparation of 6d. To a stirred suspension of Na (1.22 g, 53 mmol) in 15ml of ether under Ar was added 5d (8.5 g, 49 mmol) in 30 ml of etherover 2 h. After another 4 h of stirring a solution of I₂ (6.35 g, 25mmol) in 50 ml of ether was added dropwise over 1 h. The mixture wasleft overnight and the resulting white suspension was poured ontoether/saturated aq. NaCl. The aq. layer was extracted twice with etherand the combined organic layers were dried over MgSO₄, evaporated andchromatographed. 4.8 g (14 mmol, 56%) 6d was collected as a colourlessoil, consisting of a mixture of diastereomers and with incompletedeuteration at the two asymmetric (and acidic) carbons. ¹³C NMR(CD₃OD):209.0+208.1(—CO—), 168.5+168.2(—COO—), 53.7-53.0 (m,—CD₂—+—CHD—+—CH₂—), 51.7-50.7 (m, ester-CD₃), 25.0-23.5 (m, —CD₃).MS(ESP⁺), m/z:379 (M+39), 363 (M+23). 6a-6c were similarly prepared from5a-5c in 40-60% yield.

Preparation of 7d. NaOCOCH₃ (1.78 g, 13.0 mmol), NH₂OHxHCl (0.80 g, 11.5mmol) in 13 ml H₂O and 6d (2.80 g, 8.2 mmol.) in 35 ml CH₃COOH weremixed and stirred at 65° C. for 72 h. The mixture was cooled and most ofthe solvent was evaporated. The residue was poured onto ether/aq. NaHCO₃and the aq. layer was extracted with ether. The combined ethereal layerswere dried over Na₂SO₄, and the resulting oil was chromotographed togive 1.6 g of recovered starting material, followed by the oxime (0.025g, 0.07 mmol, 2.0%) and 7d (0.065 g, 0.19 mmol, 5.4%) as white crystals,mp. 156-158° C. ¹H NMR NMR (CD₃CN): δ 9.82 (s,1H). ¹³C NMR(CD₃CN):168.3, 136.5, 109.3, 52.2-50.9 (m, ester-CD₃), 33.5, 30.2-28.1 (m,t-CD₃). MS (ESP⁻), m/z: 334 (M−1). Similarly prepared were: 7a ¹H NMR((CD₃)₂CO): δ 9.80 (S, 1H), 3.67 (s, 6H), 1.40 (s, 18H). MS (ESP⁻), m/z:310 (M−1). 7b. ¹H NMR ((CD₃)₂CO): δ 9.80 (s, 1H), 1.40 (s, 18H). ¹³C NMR((CD₃)₂CO): δ 167.1, 135.3, 108.7, 45.8-44.7 (m, CD₃), 33.4, 29.3. MS(ESP⁻), m/z: 316 (M−1). 7c. ¹H NMR ((CD₃)₂CO): δ 9.87 (s, 1H), 3.63 (s,6H). 13C NMR ((CD₃)₂CO): δ 167.0, 135.5, 108.7, 50.9, 32.3, 29.7-28.2(m, CD₃). MS (ESP⁻), m/z: 328 (M−1).

Preparation of 3a-3d. 3a was prepared from the methyl ester 5a aspreviously described for the ethyl ester in Bull. Soc. Chim. France, 72,4330 (1970) and 3b was prepared similarly via transesterification of 5a.The extreme unwillingness of the methyl ester moiety of 3a, 6a and 7a totransesterificate and to undergo conventional hydrolysis meant thetransesterification step was performed before the dimerization step inthe preparation of 3b. The preparation of 3c and 3d started withpinacolization of hexadeuteroacetone followed by pinacol rearrangementand carboxylation to give 5c, transesterification, dimerization and ringclosure with hydroxylamine as summarised in Scheme 1 below. Theintermediate oximes could be isolated from the reaction mixture (inyields similar to those of the ring-closed products), and these wereconverted into the hydroxylamines separately under otherwise identicalconditions, or simply pooled in the next repetition of the synthesis.The hydrolysis of 7a was performed with TMSl in CdCl₃, giving thedicarboxylic acid 4 in moderate yield.

Preparation of pyrryloxyl radicals. To a degassed solution of 2 mg ofthe hydroxylamine 7a-7d in 2 ml of benzene was added ca. 10 mg NiOOH.After 5 min the suspension was filtered and the pale green blue solutionwas diluted with degassed benzene in order to obtain a solution suitablefor ESR.

Preparation of 2.5-di-t-butyl-N-hydroxy-pyrrol-3,4-dicarboxylic acid (4)

When 7a was subjected to the transesterification conditions describedabove, treated with pig liver esterase or subjected to standard alkalinehydrolysis conditions no reaction was observed. 7a (0.090 g, 0.29 mmol)was dissolved in 10 ml of dry CDCl₃ and TMSl (0.240 g, 1.20 mmol) wasadded. After heating to 55° C. overnight the mixture was diluted with 40ml of CH₂Cl₂ and washed with sat. aq. NaCl, a few drops of aq. Na₂S₂O₄in sat. aq. NaCl and finally sat. aq. NaCl. After being dried overNa₂SO₄ and evaporated the remaining solid was dissolved in heptane:ether9:1, evaporated and titurated with heptane to give the dicarboxylic acid4 (0.040 g, 0.14 mmol, 49%) as colourless crystals. ¹H NMR ((CD₃)₂CO):δ1.50 (s). ¹³C NMR ((CD₃)₂CO):δ 159.5, 141.0, 108.2, 33.7, 29.2.MS(ESP⁻), m/z: 264 (M−19). Upon treatment with NiOOH as described abovean ESR-signal with a line width almost identical to that of 3b wasrecorded.

ESR-measurements. Summarised in Table 5 are the ESR line widths for thenitroxides discussed above. All spectra were recorded at high dilutionin carefully degassed benzene at 22° C. Perdeuteration results in areduction of the line width by a factor of 2.2 for 1 and of 2.5 for 2b.For nitroxides 3a-d deuteration of the alkyl groups of the ester moietycauses a decrease by a factor of 1.9 (3c:3d) whereas deuteratin of thet-butyl groups alone has almost no influence (3a:3c). The fullydeuterated nitroxide 3d was found to have the most narrow line widthhitherto recorded for a nitroxide, 113 mG, and a nitrogen couplingconstant of 4.4 G (in benzene). The spin density distributions, nitrogencoupling constants and intrinsic line widths for 3d are compared inTable 6 to other nitroxides.

TABLE 5 ESR Line Widths of Nondeuterated and Predeuterated Nitroxides inBenzene at 23° C. Compound No. Line width/mG 1 602 1-d₁₆ 266 2b 1032 2b-d₁₃ 407 3a 228 3b 172 3c 219 3d 113

TABLE 5 ESR Line Widths of Nondeuterated and Predeuterated Nitroxides inBenzene at 23° C. Compound No. Line width/mG 1 602 1-d₁₆ 266 2b 1032 2b-d₁₃ 407 3a 228 3b 172 3c 219 3d 113

Example 7

2,2,6,6-Tetra(ethoxycarbonyl)benzo[2-d:4,5-d′]bis(1,3)dithiole

The reaction was performed under argon atmosphere using deoxygenatedsolvents. 1,2,4,5-Benzotetrathiole (1.50 g, 7.3 mmol) and K₂CO₃ (4 g)were mixed with dry DMF (70 ml) and a solution of dibromodiethylmalonate (4.26 g, 14.6 mmol) in DMF (15 ml) was added. The mixture washeated to 60° C. and stirred for 65 h. After cooling to roomtemperature, the reaction mixture was poured into ice water and thenextracted with CH₂Cl₂ (2×100 ml). The combined organic phases werewashed with water (4×50 ml), dried (Na₂SO₄) and evaporated. Yield: 3.32g (88%) ¹H MMR (CDCl₃): 6.97 (s, 2H), 4.29 (q, J=7.2 Hz, 8H), 1.28 (t,J=7.2 Hz, 12H).

Example 8

2,2,6,6-Tetra(methoxycarbonyl)-4,8-dibromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole

2,2,6,6-Tetra(ethoxycarbonyl)benzo[1,2-d:4,5-d′]bis(1,3)-dithiole (10.7g, 20.6 mmol) was dissolved in glacial acetic acid and bromine (16.5 g,0.103 mol) was added. The solution was stirred at 65° C. for 17 h andaqueous Na₂S₂O₃ was added. The aqueous slurry was extracted with CH₂Cl₂(3×100 ml), the combined organic phases were washed with water (3×50ml), dried (MgSO₄) and evaporated. The residue was triturated with CH₃CNand dried. Yield: 10.1 g (72%).

¹H NMR (DMSO-d₆): 4.28 (q, J=7.2 Hz, 8H), 1.21 (t, J=7.2 Hz, 12H).

Example 9

4,8-Dibromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,6-dispiro-(4,4-dimethyl-3,5-dioxane)

2,2,6,6-Tetra(methoxycarbonyl)-4,8-dibromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole(6.76 g, 10.0 mmol) was dissolved in dry THF, the solution was cooled to0° C. and a solution of DIBAL in toluene (17.8 ml, 100 mmol) was addeddropwise. The solution was heated to reflux temperature for 3 h and thenallowed to cool to room temperature. Methanol (20 ml) was added dropwisefollowed by water (60 ml) and the pH was adjusted to 2 using aqueous 6 MHCl. The solvents, except water, were removed by evaporation and theprecipitate was collected by filtration. The product was washed withwater, acetonitrile, dried and then suspended in dry acetone (600 ml).BF₃·Et₂O (2.52 ml, 20 mmol) was added and the solution was stirred for20 min. Solid K₂CO₃ (6.0 g) was added and stirring was continued foranother 5 min. After filtering through a short pad of basic alumina, thesolvents were removed by evaporation, the residue was triturated withCH₂Cl₂ and dried. Yield: 1.12 g (19%)

¹H NMR (DMSO-D₆): 4.15 (S, 8H), 1.37 (S, 12H).

Example 10

4-Bromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,6-dispiro-(4,4-dimethyl-3,5-dioxane)

4,8-Dibromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,6-dispiro-(4,4-dimethyl-3,5-dioxane)(1.14 g, 1.94 mmol) was dissolved in dry THF (270 ml) under anatmosphere of argon. After cooling the solution to −45° C., a solutionof n-BuLi in hexane (2.5M, 2.02 mmol) was added dropwise. After stirringfor 5 min, methanol (3 ml) was added, the solution was allowed to attainroom temperature and the solvents were evaporated. The product waspurified by chromatography on silica gel using a mixture of CH₂Cl₂ andmethanol (99.5:0.5) as the eluent. Yield: 0.70 g (71%).

¹H NMR (CDCl₃): 6.80 (s, 1H), 4.15 (s, 8H), 1.47 (s, 12H).

Example 11

Tris(benzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl-2,6-dispiro-(4,4-dimethyl-3,5-dioxane))methanol

4-Bromobenzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,6-dispiro-(4,4-dimethyl-3,5-dioxane)(0.99 g, 1.94 mmol) was suspended in dry diethyl ether (28 ml) under anatmosphere of argon. A solution of n-BuLi (2.5M in hexane, 1.94 mmol)was added dropwise and, after 5 min, a solution of diethyl carbonate0.078 ml, 0.64 mmol) in diethyl ether (3 ml) was added slowly. Afterstirring for 18 h, ethanol (5 ml) was added and the solvent was removedby evaporation. The product was purified by chromatography on silica gelusing a mixture of CHCl₃ and ethyl acetate (20:1) as the eluent. Yield:0.65 g (76%)

¹H NMR (CDCl₃): 7.16 (s, 3H), 6.01 (s, 1H), 3.86-4.22 (m, 24H), 1.43,1.41, 1.37, 1.32 (4s, 36H).

Example 12

Tris(8-ethoxycarbonylbenzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl-2,6-dispiro-(4,4dimethyl-3,5-dioxane))methanol

Tris(benzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl-2,6-dispiro-(4,4-dimethyl-3,5-dioxane))methanol(0.205 g, 0.156 mmol) was dissolved in dry benzene (12 ml) containingN,N N′,N′-tetramethylethylene diamine (0.33 ml, 2.18 mmol) under anatmosphere of argon. A solution of t-BuLi in pentane (1.5M, 2.18 mmol)was added dropwise and stirring was continued for 40 min. The solutionwas then transferred into another flask, kept at 0° C. and containingdiethylpyrocarbonate (1.3 ml, 8.82 mmol) and benzene (6 ml). Afterstirring for 45 min, an aqueous NaH₂PO₄ buffer was added, the organicphase was separated, washed with water and evaporated. The product waspurified by preparative HPLC. Yield: 55 mg (23%).

¹H NMR (CDCl₃): 6.68 (s, 1H), 4.41-4.52 (m, 6H), 3.86-4.21 (m, 24 H),1.22-1.60 (m, 45H).

Example 13

Tris(8-ethoxycarbonyl-2,2,6,6-tetrahydroxymethylbenzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl)methanol

Tris(8-ethoxycarbonylbenzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl-2,6-dispiro-(4,4-dimethyl-3,5-dioxane))methanol(55 mg, 0.0359 mmol) was dissolved in a mixture of glacial acetic acid(20 ml) and water (5 ml) and the solution was stirred at roomtemperature for 42 h. The solvents were removed by evaporation, tracesof acid were removed by addition of benzene followed by evaporation.HPLC analysis indicated >98 purity of the product. Yield: 42.4 mg (91%).

MS (ESP⁻, m/e): 1293 (M⁺, 68%), 1291 ([M−2]⁻, 100%).

Example 14

Tris(8-carboxy-2,2,6,6-tetrahydroxymethylbenzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl)methylsodium salt

Tris(8-ethoxycarbonyl-2,2,6,6-tetrahydroxymethylbenzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl)methanol (3.4 mg, 0.0026 mmol) wasdissolved in acetonitrile (2 ml) and the solution was cooled to 0° C.Trifluoromethane-sulfonic acid (0.017 ml) was added and after 15 min, asolution of SnCl₂ (0.4 mg) in acetonitrile (1 ml) was added. Afteranother 15 min, an aqueous NaH₂PO₄ buffer was added and the solventswere removed by evaporation. The residue was suspended in water and thepH was adjusted to 12 using an 1M aqueous NaOH solution. After stirringfor 1 h, the solution was neutralized with 1M aqueous HCl and thesolvent was removed by evaporation. The product was purified bypreparative HPLC. Yield: 2.0 mg (60%).

ESR (1.5 mM in H₂O, 100 G): singlet, linewidth 100 mG.

This compound is also useful in the method of the invention.

Example 15

2,2,6,6-Tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole-4-carboxylic acid

2,2,6,6-Tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole (10.0 g, 45.0mmol; prepared according to WO-91/12024) was dissolved in dry THF (200mL) under an argon atmosphere. The solution was cooled to −20° C. andn-butyllithium (20.0 mL, 50.0 mmol) in hexane was added. After attainingambient temperature, the reaction mixture was transferred onto solidcarbon dioxide (150 g) and allowed to stand overnight. Water (200 mL)was added and pH was adjusted to 10 using 2M aqueous NaOH. After washingwith ether, the aqueous phase was acidified with 2M hydrochloric acid topH 2 and extracted with ether (2*300 mL). The organic phases were dried(Na₂SO₄) and evaporated to give the pure product.

Yield: 10.7 g (89%).

1H NMR (CDCl3, 300 MHz) δ: 6.50 (s, 1H), 1.71 (s, 12H).

¹³C NMR (CDCl₃, 75 MHz) δ: 165.1, 140.9, 140.8, 119.8, 98.9, 97.3, 25.6.

Example 16

2,2,6,6-Tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole-4-carboxylic acidmethyl ester

2,2,6,6-Tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole-4-carboxylic acid(10.0 g, 38.0 mmol) was dissolved in dry DMF (100 mL). Potassiumcarbonate (15.2 g, 110.0 mmol) was added and the reaction was heated to55° C. for 30 min. After cooling to ambient temperature, methyl iodide(15.6 g, 110.0 mmol) was added and the solution was stirred overnight.The precipitate was filtered off and the solution was evaporated. Theresidue was dissolved in saturated aqueous NaHCO₃ and ether. The aqueouslayer was discarded and the organic phase was dried (Na₂SO₄), filteredand evaporated to give 9.4 g (88%) of the pure product.

¹H NMR (CDCl₃, 300 MHz) δ: 6.44 (s, 1H), 3.85 (s, 3H), 1.65 (s, 12H).

¹³C NMR (CDCl₃, 75 MHz) δ: 163.4, 140.8, 140.6, 119.0, 99.9, 99.4, 51.9,25.6.

Example 17

Bis-(2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)diyhiole-4-yl)-mono-(22,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole-4-yl)methanol

2,2,6,6-Tetramethylbenzo[1,2-d:4,5-d ′]bis(1,3)dithiole (2.86 g, 10mmol; prepared according to WO-91/12024) was dissolved in anhydrous THF(75 mL) and cooled to −70° C. n-Butyllithium (4.4 mL, 2.5M in hexane)was added. The reaction mixture was allowed to reach ambienttemperature.4-Methoxycarbonyl-2,2,6,6-tetramethylbenzo-[1,2-d:4,5-d′]-bis-(1,3)-dioxole(1.4 g, 5 mmol) was added as a solid. After 1 hour, the mixture wasquenched with saturated aqueous NaH₂PO₄. The aqueous phase was discardedand the organic layer evaporated. The residue was dissolved indichloromethane, washed with water and dried (Na₂SO₄). The product waspurified by column chromatography (dichloromethane:heptane, 1:1) giving1.8 g (44%) of pure product.

¹H NMR (CDCl₃, 300 MHz) δ: 7.10 (broad s, 2 H, ArH), 6.39 (s, 1 H, ArH),4.79 (s, 1 H, OH), 1.82-1.56 (m, 24 H, CH₃), 1.53 (s, 6 H, CH₃), 1.46(s, 6 H, CH₃).

Example 18

Bis-(B-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dithiole-4-yl)-mono-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methanol

Bis-(2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dithiole-4-yl)-mono-(2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methanol (0.50 g, 0.61 mmol) wasdissolved in dry benzene (6.0 mL) under an atmosphere of argon.t-Butyllitium (2.44 mL, 1.5M in pentane) and TMEDA (0.545 mL, 3.66 mmol)were added. The reaction mixture was subjected to ultrasound for 25 min.and was then slowly added to a solution of diethyl carbonate (7.2 mL,59.4 mmol) in dry benzene (16 mL). After stirring for 1.5 h, aqueousNaH₂PO₄(50 mL) was added. The organic layer was separated, washed withwater, dried (Na₂SO₄) and evaporated. After purification by preparativeHPLC 130.0 mg (21%)) of the pure product was obtained.

¹H NMR (CDCl₃, 300 MHz) δ: 4.98 (s, 1H), 4.28-4.37 (m, 6H) 1.48-1.79 (m,36H), 1.46 (t, 6H, J 7.0 Hz), 1.38 (t, 3H, J 7.0 Hz).

¹³C NMR (CDCl₃, 75 MHz) δ: 166.2, 166.0, 162.9, 141.9, 141.6, 141.2,140.8, 140.4, 140.0, 136.6, 134.5, 129.9, 128.5, 128.1, 127.8, 127.2,120.3, 118.9, 111.9, 101.1, 80.6, 62.1, 61.0, 60.3, 60.2, 59.8, 59.2,34.4, 34.3, 33.5, 28.8, 28.1, 27.0, 26.9, 26.5, 25.8.

Example 19

Bis-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dithiole-4-yl)-mono-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methyl

Bis-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dithiol-4-yl)-mono-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methanol(520 mg, 0.501 mmol) was dissolved in dry degassed dichloromethane (15mL) together with tin(II) chloride (95 mg, 0.501 mmol) and acetonitrile(5 mL). BF₃.Et₂O (70 μL, 0.557 mmol) was added and the solution wasstirred for 20 min. After addition of dichloromethane (80 ML) andwashing with degassed water (80 mL), the organic layer was separated,dried (MgSO₄), filtered and evaporated. The product was purified bypreparative HPLC. Yield: 110 mg (22%).

ESR (THF, 200 G) singlet, line width 325 mG.

Overhauser enhancement (THF, 2.1 mM): !156 at 4 W microwave power.

Stability measurements: Half life in acetonitrile without exclusion orair: 2000 h.

Example 20

Bis-(8-potassium carboxylate-2,2,6,6-tetramethylbenzo [1,2-d:45-d′]-bis(1,3)dithiol-4-yl)-mono-(8-potassiumcarboxylate-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methyl

Bis-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dithiol-4-yl)-mono-(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl)methyl(132 mg, 0.129 mmol) was dissolved in ethanol (10 mL). Aqueous potassiumhydroxide (5 mL, 1.0M) was added and the reaction mixture was stirred at50° C. overnight. After evaporation of the ethanol, the mixture wasstirred for 1 h at 50° C. and was then acidified with 2M hydrochloricacid. The aqueous phase was extracted with ether. The organic phase wasseparated, dried (MgSO₄) filtered and evaporated. The product waspurified by preparative HPLC. The fractions were evaporated and waterwas added. The aqueous layer was extracted with ether. The organic layerwas separated, dried (MgSO₄), filtered and evaporated. The product wasdissolved by adding water and 1M KOH (0.387 mL, 0.387 mmol). Thesolution was lyophilized.

Yield: 101 mg (75%).

ESR (H₂O, 200 G): singlet, line width 105 mG. Overhauser enhancement(H₂O, 6.9 mM): 219 at 0.012 W microwave power.

Example 21

Benzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,2,6,6-tetracarboxylic acidtetraethyl ester

1,2,4,5-benzenetetrathiol (1.50 g, 7.28 mmol) was dissolved in dry DMF(55 ml) under an atmosphere of Argon and K₂CO₃ (4.0 g) was addedtogether with 2,2-dibromomalonate ethyl ester (4.26 g, 14.6 mmol). Thesolution was stirred at room temperature for 16 h and then at 60° C. foran additional 5 h. The reaction mixture was then poured into anice-water mixture (200 g-200 ml) and extracted with ethyl acetate (2×250ml). The combined organic phases were washed with water (4×100 ml) dried(Na₂SO₄) and evaporated. The crude product was washed sufficiently pureto be used in the next step without purification. Yield: 3.05 g (80%) 1HNMR (300 MHz, CDCl₃): 6.91 (s,2H), 4.29 (q, J=7.2 Hz, 8H), 1.28 (t,J=7.2 Hz, 12H).

Example 22

2,2,6,6-tetra(hydroxymethyl-d₂)benzo[1,2-d:4,5-d′]bis(1,3)dithiole

A dry Soxhlet setup was provided withBenzo[1,2-d:4,5-d′]bis(1,3)dithiole-2,2,6,6-tetracarboxylic acidtetraethyl ester (5.0 g, 9.65 mmol) in the upper compartment and amixture of lithium aluminium deuteride (1.62 g, 38.6 mmol) and diethylether (300 ml) in the lower, round-bottomed flask. The ether was heatedto reflux temperature for 20 h and the mixture was then allowed to cool.Methanol (150 ml) was added dropwise by water (50 ml). The mixture wasacidified with concentrated HCl (20 ml) and the solvent was reduced to50 ml by evaporation in vacuum. The white solid was filtered off, washedwith water (2×25 ml) and dried. Yield 3.15 g (91%).

1NMR (300 MHz, DMSO-d₆): 7.06 (2,2H), 5.45 (br s, 4H)

Example 23

2,2,6,6-Tetra(dimetylthexylsilyloxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiole

The reaction was performed under argon atmosphere.2,2,6,6-Tetra(hydroxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiole (0.8 g,2.2 mmol) was dissolved in DMF (20 mL). Imidazole (1.1 g, 15.8 mmol) wasadded and the solution was cooled to 0° C. Dimethylthexylsilyl chloride(2.8 g, 15.8 mmol) was added dropwise (ca 2 min). The solution wasstirred for 48 hours at ambient temperature. The reaction mixture waspoured into ice/water, CH₂Cl₂(100 mL) was added and the two phases wereseparated. The organic phase was washed with 1M HCl and water (3*100mL). The solution was dried (Na₂SO₄) and evaporated. The product waspurified by column chromatography using dichloromethane-heptane (1:9) aseluent.

Yield: 1.1 g (52%)

¹H NMR (CDCl₃, 300 MHz) δ: 6.84 (s, 2H, ArH), 3.94 (s, 8H, CH₂), 1.62(septet, 4H, J 6.8 Hz, CH), 0.88 (d, 24H, J 6.8 Hz, CH₃), 0.84 (s, 24H,CH₃), 0.08 (s, 24H, Si(CH₃)₂).

¹³C NMR (CDCl₃, 75 MHz) δ: 134.3, 115.8, 74.2, 65.0, 34.2, 25.1, 20.3,18.6, −3.6.

Example 24

Bis(2,2,6,6-tetra(dimetylthexylsilyloxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methanol

The reaction was performed under argon atmosphere.2,2,6,6-Tetra(dimetylthexylsilyloxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiole(7.0 g, 7.6 mmol) was dissolved in dry THF (50 mL). The solution wascooled to −70° C. n-Butyllithium (5.0 mL, 1.6M in hexane) was added andthe temperature was allowed to attain ambient temperature and wasstirred for 1 h. The solvent was evaporated in vacuum at ambienttemperature and diethyl ether (20 mL) was added. Then,4-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxole(0.8 g, 2.9 mmol) was added in one portion and the reaction mixture wasstirred at ambient temperature for 12 h. The mixture was poured into aNaH₂PO₄ solution, the phases were separated and the aqueous phase wasextracted with diethyl ether (2*100 mL). The organic phases were dried(Na₂SO₄) and evaporated. The residue was purified by preparative HPLC.

Yield: 3.7 g (62%).

¹H NMR (CDCl₃, 300 MHz) δ: 6.80 (s, 2H, ArH), 6.26 (s, 1H, ArH), 4.95(s, 1H, OH), 3.8 (br m, 16H, CH₂), 1.5 (br m, 20H, CH₃+CH), 0.9 (d, 48H,CH₃), 0.7 (s, 48H, CH₃), 0.2 (2 s, 48H, Si(CH3)₂).

¹³C NMR (CDCl₃, 75 MHz) δ: 141.5, 140.3, 139.8, 139.6, 131.7, 118.6,117.1, 108.1, 94.4, 80.0, 65.4, 34.1, 25.9, 25.0, 20.3, 18.7, −3.2.

Example 25

Bis(8-ethoxycarbonyl-2,2,6,6-tetra(hydroxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methanol

Bis(2,2,6,6-tetra(dimetylthexylsilyloxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methanol(3.2 g, 1.54 mmol) was dissolved in heptane (12.8 mL) and dry benzene(10.7 mL) together with TMEDA (3.2 mL, 21.6 mmol) under an atmosphere ofargon. The solution was cooled to −22° C. and t-BuLi (14.4 mL, 1.5M inpentane) was added. After stirring for 3 h at −22° C., the reactionmixture was transferred into a solution of diethyl pyrocarbonate (12.8mL, 87 mmol) in heptane (23 mL) and dry benzene (23 mL) which was keptat −22° C. The reaction mixture was then allowed to attain ambienttemperature. After stirring for an additional hour, a saturated aqueoussolution of NaH₂PO₄ (40 mL) was added. The mixture was stirred for onehour, the organic phase was separated, washed with water (2*100 mL) andacetonitrile (2*100 mL). The heptane/benzene phase was evaporated andthen dissolved in THF (25 mL) A solution of Bu₄NF in THF (20 mL, 20mmol) was added and the mixture was stirred overnight. After evaporationof the solvent, the residue was partitioned between water (300 mL) andethyl acetate (300 mL). The organic phase was washed with water (2*100mL), dried (Na₂SO₄) and evaporated. Purification by preparative HPLCgave 400 mg (22%) pure product.

¹H NMR (CDCl₃, 300 MHz) δ: 5.78-5.92 (m, 6H), 5.03-5.52 (m, 24H),2.98-3.21 (m, 12H), 2.90 (t, 6H, J 7.0 Hz), 2.84 (t, 3H, J 6.9 Hz).

Example 26

Bis(8-ethoxycarbonyl-2,2,6,6-tetra(hydroxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methyl

Bis(8-ethoxycarbonyl-2,2,6,6-tetra(hydroxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methanol(294 mg, 0.25 mmol) was dissolved in acetonitrile (70 mL) under anatmosphere of argon. After cooling to 0° C., trifluoromethane sulfonicacid (190 μL, 2.2 mmol) was added. After stirring for 3 min, tin(II)chloride (48 mg, 0.25 mmol) dissolved in acetonitrile (7 mL) was added.After 1 min, a saturated aqueous solution of NaH₂PO₄ (50 mL) was added.The aqueous phase was washed with acetonitrile (2*50 mL), the combinedorganic phases were dried (Na₂SO₄) and evaporated. Purification bypreparative HPLC gave 176 mg (61%) of the pure product.

ESR (H₂O, 200 G): singlet, linewidth 433 mG.

Example 27

Bis(8-carboxy-2,2,6,6-tetra(hydroxymethyl)benzo[2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(8-carboxy-2,2,6,6-tetramethylbenzo[2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methyl sodium salt

Bis(8-ethoxycarbonyl-2,2,6,6-tetra(hydroxymethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiol-4-yl)-mono(8-ethoxycarbonyl-2,2,6,6-tetramethylbenzo[2-d:4,5-d′]-bis(1,3)dioxol-4-yl))methyl(316 mg, 0.275 mmol) was dissolved in a mixture of 1M aqueous NaOH (3mL), water (1.5 mL) and ethanol (3 mL). The solution was stirred atambient temperature for 15 min, the ethanol was removed by evaporation,and the residue was stirred at ambient temperature for additional 2hours. After evaporation to near dryness, the pure acid (240 mg, 82%)was isolated by preparative HPLC followed by lyophilization. The acidwas converted into the corresponding sodium salt by the addition ofwater (50 mL) followed by adjustment of the pH to 7 with 1M aqueous NaOHand lyophilization.

ESR (3.4 mM in H₂O, 200 G): singlet, linewidth 120 mG.

Overhauser enhancement (aqueous solution as above): 164 at 5 W microwavepower.

Stability measurements: Half life in water without exclusion of air: 120h.

Example 28

A schematic representation of the “elementary method” according to theinvention using the perdeuterated hydroxy trityl. The self- and oxygenbroadening is given by eq. 1, the inhomogeneous broadening is ΔH_(PP)^(G)=60 mG and the relaxivity 0.4 mM⁻¹s⁻¹. Images A, B and C:

$I_{A} = {A\left\{ {{0.4\quad {mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{{\exp \left( {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right)}\frac{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{A}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H^{\prime}}}}}} \right\}} - 1} \right\}}$

$I_{B} = {A\left\{ {{0.4\quad {mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{\exp \left\{ {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right\} \frac{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{B}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H^{\prime}}}}}} \right\}} - 1} \right\}}$

$I_{C} = {A \times \left\{ {{0.4\quad {mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{\exp \left\{ {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right\} \frac{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{C}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H^{\prime}}}}}} \right\}} - 1} \right.}$

$c_{rad},{{\Delta \quad H_{pp}^{L}} = {\frac{2}{\sqrt{3}\gamma_{e}T_{2e}}\quad {and}\quad {\alpha\gamma}_{e}T_{1e}}}$

Example 29

A schematic representation of the preferred method according to theinvention using the perdeuterated hydroxy trityl. The self- and oxygenbroadening is given by eq. 1, the inhomogeneous broadening is ΔH_(PP)^(G)=60 mG and the relaxivity 0.4 mM⁻¹s⁻¹. Images A,B,C,D and E:

$\frac{I_{A}}{I_{E}} = {A\left\{ {0.4{mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{{\exp \left( {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right)}\frac{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{A}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H}}}}} \right.} \right.}$

$\frac{I_{B}}{I_{E}} = {A\left\{ {0.4{mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{\exp \left\{ {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right\} \frac{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{H^{\prime 2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{B}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H}}}}} \right.} \right.}$

$\frac{I_{C}}{I_{E}} = {A \times \left\{ {0.4{mM}^{- 1}s^{- 1}c_{rad}\left\{ {1 - {\sqrt{\frac{2}{\pi}}\frac{1}{90\quad {mG}}{\int_{- \infty}^{\infty}{\exp \left\{ {{- 2}{H^{\prime 2}/\left( {60\quad {mG}} \right)^{2}}} \right\} \frac{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}}}{1 + {\frac{4}{3}{\left( {{\Delta \quad H} - H^{\prime}} \right)^{2}/\Delta}\quad H_{pp}^{L^{2}}} + {\frac{2}{\sqrt{3}}\alpha \quad P_{C}\gamma_{e}{T_{1e}/\Delta}\quad H_{pp}^{L}}}{H}}}}} \right.} \right.}$

$c_{rad},{{\Delta \quad H_{pp}^{L}} = {\frac{2}{\sqrt{3}\gamma_{e}T_{2e}}\quad {and}\quad {\alpha\gamma}_{e}T_{1e}}}$

What is claimed is:
 1. A method of determining oxygen concentration in asample, said method comprising the following steps: introducing intosaid sample an effective amount of a physiologically tolerable freeradical having an esr transition with a linewidth measured in water ofless than 400 mG; irradiating said sample with radiation of an amplitudeand frequency selected to stimulate an electron spin resonancetransition of said radical; detecting electron spin resonance enhancedmagnetic resonance signals from said sample under at least first, secondand third conditions, wherein under said first and second conditionssaid radiation is of a first frequency, under said third conditions saidradiation is of a second frequency different from said first frequency,under said first, second and third conditions said radiation is of afirst, second and third amplitude, said first and second amplitudes atleast being different from each other; and manipulating said detectedsignals to determine oxygen concentration in said sample.
 2. A method asclaimed in claim 1 wherein the step of manipulating said detectedsignals comprises generating an image data set.
 3. A method as claimedin claim 2 comprising (a) generating a first OMRI image of said sampleat VHF power P_(A), irradiation period T_(VHF1) and on-resonance (ΔH=0),(b) generating a second OMRI image of said sample at a second VHF powerP_(B), irradiation time T_(VHF1) and on-resonance (ΔH=0) (c) generatinga third OMRI image of said sample at VHF power P_(C), irradiation periodT_(VHF1) and off-resonance (ΔH≠0) (d) manipulating the images obtainedin steps (a) to (c) and calibrating using parameters determined ex vivoto provide an oxygen image of said sample.
 4. A method as claimed inclaim 3 wherein additionally a fourth image is generated at VHF powerP_(A) and irradiation period T_(VHF2) and a fifth MR image is generatedwithout VHF irradiation.
 5. A method as claimed in claim 1 comprisingthe additional step of generating a native MR image of the sample.
 6. Amethod as claimed in claim 1 in which the step of manipulating saiddetected signals comprises fitting the measured degree of saturation ofthe esr transition to a Voigtian function.
 7. A method as claimed inclaim 1 wherein said physiologically tolerable free radical is a radicalwhich distributes into the extracellular fluid.
 8. A method as claimedin claim 1 wherein said physiologically tolerable free radical has anesr transition with a linewidth measured in water of less than 150 mG.9. A method as claimed in claim 8 wherein said radical has an esrtransition with a linewidth of less than 60 mG.
 10. A method as claimedin claim 1 wherein said physiologically tolerable free radical is atrityl.
 11. A method as claimed in claim 10 wherein said trityl is offormula

wherein: n is 0, 1, 2 or 3; R¹ is a carboxyl group or a derivativethereof; R² is an optionally hydroxylated C₁₋₆-alkyl group; preferably aC^(n)H₃ or C^(n)H₂OH group (where n is 1 or 2 i.e. ²H is deuterium); andthe salts and precursors and deuterated analogs thereof.
 12. A method asclaimed in claim 10 wherein said trityl is of formula:


13. The method as claimed in claim 1, wherein manipulating said detectedsignals includes calibrating using parameters determined ex vivo. 14.The method as claimed in claim 13, wherein said parameters aredetermined under substantially the same conditions existing in thesample whose oxygen concentration is to be determined.
 15. The method asclaimed in claim 14, wherein said parameters are determined for a rangeof oxygen and radical concentrations in a fluid sample which correspondsto the fluid in which oxygenation is to be determined, at the sametemperature as the temperature of the fluid in which oxygenation is tobe determined.
 16. The method as claimed in claim 14, wherein saidparameters are determined in blood or a biological fluid correspondingto the fluid in which oxygenation is to be determined.
 17. The method asclaimed in claim 14, wherein said parameters are determined at 37° C.18. The method as claimed in claim 13, wherein said parameters aredetermined under anaerobic conditions.
 19. The method as claimed inclaim 13, wherein said parameters are determined at an oxygen partialpressure of 100 mmHg.
 20. The method as claimed in claim 13, whereinsaid parameters are determined for a range of oxygen concentrations upto a concentration of 0.5 mM.
 21. The method as claimed in claim 20,wherein said parameters are determined for a range of oxygenconcentrations up to a concentration of 0.1 mM.
 22. The method asclaimed in claim 13, wherein said parameters are determined for a rangeof radical concentrations up to a concentration of 1.5 mM.
 23. Themethod as claimed in claim 22, wherein said parameters are determinedfor a range of radical concentrations up to a concentration of 1.0 mM.24. The method as claimed in claim 22, wherein said parameters aredetermined for a range of radical concentrations up to a concentrationof 0.2 mM.